[NMR paper] Biomass production of site selective 13C/15N nucleotides using wild type and a transketolase E. coli mutant for labeling RNA for high resolution NMR.
Journal of biomolecular NMR (2012). Volume: 52, Issue: 2. Pages: 103-14. Chandar S Thakur, Yiling Luo, Bin Chen, Nadukkudy V Eldho, T Kwaku Dayie et al.
Characterization of the structure and dynamics of nucleic acids by NMR benefits significantly from position specifically labeled nucleotides. Here an E. coli strain deficient in the transketolase gene (tktA) and grown on glucose that is labeled at different carbon sites is shown to facilitate cost-effective and large scale production of useful nucleotides. These nucleotides are site specifically labeled in C1' and C5' with minimal scrambling within the ribose ring. To demonstrate the utility of this labeling approach, the new site-specific labeled and the uniformly labeled nucleotides were used to synthesize a 36-nt RNA containing the catalytically essential domain 5 (D5) of the brown algae group II intron self-splicing ribozyme. The D5 RNA was used in binding and relaxation studies probed by NMR spectroscopy. Key nucleotides in the D5 RNA that are implicated in binding Mg(2+) ions are well resolved. As a result, spectra obtained using selectively labeled nucleotides have higher signal-to-noise ratio compared to those obtained using uniformly labeled nucleotides. Thus, compared to the uniformly (13)C/(15)N-labeled nucleotides, these specifically labeled nucleotides eliminate the extensive (13)C-(13)C coupling within the nitrogenous base and ribose ring, give rise to less crowded and more resolved NMR spectra, and accurate relaxation rates without the need for constant-time or band-selective decoupled NMR experiments. These position selective labeled nucleotides should, therefore, find wide use in NMR analysis of biologically interesting RNA molecules.
[NMR paper] Biomass production of site selective 13C/15N nucleotides using wild type and a transketolase E. coli mutant for labeling RNA for high resolution NMR.
From Mendeley Biomolecular NMR group:
Biomass production of site selective 13C/15N nucleotides using wild type and a transketolase E. coli mutant for labeling RNA for high resolution NMR.
Journal of biomolecular NMR (2012). Volume: 52, Issue: 2. Pages: 103-14. Chandar S Thakur, Yiling Luo, Bin Chen, Nadukkudy V Eldho, T Kwaku Dayie et al.
Characterization of the structure and dynamics of nucleic acids by NMR benefits significantly from position specifically labeled nucleotides. Here an E. coli strain deficient in the transketolase gene (tktA) and grown on glucose that is labeled at...
nmrlearner
Journal club
0
10-12-2012 09:58 AM
[NMR paper] Biomass production of site selective 13C/15N nucleotides using wild type and a transketolase E. coli mutant for labeling RNA for high resolution NMR.
From Mendeley Biomolecular NMR group:
Biomass production of site selective 13C/15N nucleotides using wild type and a transketolase E. coli mutant for labeling RNA for high resolution NMR.
Journal of biomolecular NMR (2012). Volume: 52, Issue: 2. Pages: 103-14. Chandar S Thakur, Yiling Luo, Bin Chen, Nadukkudy V Eldho, T Kwaku Dayie et al.
Characterization of the structure and dynamics of nucleic acids by NMR benefits significantly from position specifically labeled nucleotides. Here an E. coli strain deficient in the transketolase gene (tktA) and grown on glucose that is labeled at...
nmrlearner
Journal club
0
08-24-2012 08:01 PM
Biomass production of site selective 13C/15N nucleotides using wild type and a transketolase E. coli mutant for labeling RNA for high resolution NMR
Biomass production of site selective 13C/15N nucleotides using wild type and a transketolase E. coli mutant for labeling RNA for high resolution NMR
Abstract Characterization of the structure and dynamics of nucleic acids by NMR benefits significantly from position specifically labeled nucleotides. Here an E. coli strain deficient in the transketolase gene (tktA) and grown on glucose that is labeled at different carbon sites is shown to facilitate cost-effective and large scale production of useful nucleotides. These nucleotides are site specifically labeled in C1â?² and C5â?² with...
nmrlearner
Journal club
0
11-30-2011 10:45 PM
A simple protocol for amino acid type selective isotope labeling in insect cells with improved yields and high reproducibility
A simple protocol for amino acid type selective isotope labeling in insect cells with improved yields and high reproducibility
Abstract An easy to use and robust approach for amino acid type selective isotope labeling in insect cells is presented. It relies on inexpensive commercial media and can be implemented in laboratories without sophisticated infrastructure. In contrast to previous protocols, where either high protein amounts or high incorporation ratios were obtained, here we achieve both at the same time. By supplementing media with a well considered amount of yeast extract,...
nmrlearner
Journal club
0
10-05-2011 08:57 PM
Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle.
Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle.
Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle.
J Biomol NMR. 2010 Dec;48(4):179-92
Authors: Thakur CS, Sama JN, Jackson ME, Chen B, Dayie TK
Escherichia coli (E. coli) is an ideal organism to tailor-make labeled nucleotides for biophysical studies of RNA. Recently, we showed that adding labeled formate enhanced the isotopic enrichment at protonated carbon...
nmrlearner
Journal club
0
03-01-2011 12:14 PM
Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli
Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle
Abstract Escherichia coli (E. coli) is an ideal organism to tailor-make labeled nucleotides for biophysical studies of RNA. Recently, we showed that adding labeled formate enhanced the isotopic enrichment at protonated carbon sites in nucleotides. In this paper, we show that growth of a mutant E. coli strain DL323 (lacking succinate and malate dehydrogenases) on 13C-2-glycerol and 13C-1,3-glycerol enables selective labeling at many useful sites for RNA NMR...
nmrlearner
Journal club
0
11-09-2010 03:17 PM
Site-specific labeling of nucleotides for making RNA for high resolution NMR studies
Abstract Escherichia coli (E. coli) is a versatile organism for making nucleotides labeled with stable isotopes (13C, 15N, and/or 2H) for structural and molecular dynamics characterizations. Growth of a mutant E. coli strain deficient in the pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase (K10-1516) on 2-13C-glycerol and 15N-ammonium sulfate in Studier minimal medium enables labeling at sites useful for NMR spectroscopy. However, 13C-sodium formate combined with 13C-2-glycerol in the growth media adds labels to new positions. In the absence of labeled formate, both C5 and...
nmrlearner
Journal club
0
08-14-2010 04:19 AM
High Resolution 1H Detected 1H,13C Correlation Spectra in MAS Solid-State NMR using Deuterated Proteins with Selective 1H,2H Isotopic Labeling of Methyl Groups
High Resolution <SUP>1</SUP>H Detected <SUP>1</SUP>H,<SUP>13</SUP>C Correlation Spectra in MAS Solid-State NMR using Deuterated Proteins with Selective <SUP>1</SUP>H,<SUP>2</SUP>H Isotopic Labeling of Methyl Groups
Vipin Agarwal, Anne Diehl, Nikolai Skrynnikov, and Bernd Reif
J. Am. Chem. Soc.; 2006; 128(39) pp 12620 - 12621;
Abstract:
MAS solid-state NMR experiments applied to biological solids are still hampered by low sensitivity and resolution. In this work, we employ a deuteration scheme in which individual methyl groups are selectively protonated. This labeling scheme...
Biomass production of site selective 13C/15N nucleotides using wild type and a transketolase E. coli mutant for labeling RNA for high resolution NMR.
Linear Mode
